1. Patent Search
  2. Details

METHOD FOR PRODUCING TREHALOSE EMPLOYING A TREHALOSE PHOSPHORYLASE VARIANT

20210087598 ยท 2021-03-25

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to a method for producing trehalose, comprising the steps of mixing and reacting, in any order, (i) at least one alpha-phosphorylase capable of catalyzing the production of alpha-D-glucose 1-phosphate intermediate from a saccharide raw material, and from at least one phosphorus source; (ii) at least one trehalose phosphorylase capable of catalyzing the production of trehalose from an alpha-D-glucose 1-phosphate intermediate and a glucose substrate, wherein the trehalose phosphorylase is a trehalose phosphorylase variant with an amino acid sequence which differs from the amino acid sequence of a wild type trehalose phosphorylase in at least one amino acid position, (iii) at least one saccharide raw material which produces an alpha-D-glucose 1-phosphate intermediate and a co-product by catalytic action of the alpha-phosphorylase; and (iv) at least one phosphorus source selected from the group consisting of a phosphoric acids and an inorganic salt thereof.

    Claims

    1. A method for producing trehalose, comprising the steps of mixing and reacting, in any order, a) at least one alpha-phosphorylase capable of catalyzing the production of alpha-D-glucose 1-phosphate intermediate from a saccharide raw material selected from the group consisting of sucrose and starch, and from at least one phosphorus source selected from the group consisting of a phosphoric acid and an inorganic salt thereof; b) at least one trehalose phosphorylase capable of catalyzing the production of trehalose from an alpha-D-glucose 1-phosphate intermediate and a glucose substrate, wherein the trehalose phosphorylase is a trehalose phosphorylase variant with an amino acid sequence which differs from the amino acid sequence of a wild type trehalose phosphorylase in at least one amino acid position; c) at least one saccharide raw material selected from the group consisting of sucrose and starch which produces an alpha-D-glucose 1-phosphate intermediate and a co-product selected from the group of fructose co-product and starch co-product by catalytic action of the alpha-phosphorylase; and d) at least one phosphorus source selected from the group consisting of a phosphoric acids and an inorganic salt thereof.

    2. The method of claim 1, further comprising the step of mixing and reacting, in any order, i) a glucose substrate; and/or ii) at least one glucose isomerase with the at least one alpha-phosphorylase, the at least one trehalose phosphorylase, the at least one saccharide raw material selected from the group consisting of sucrose and starch, and the at least one phosphorus source.

    3. The method of claim 1, wherein the method is characterized by two or more of the following conversions selected from the group consisting of: a) the conversion of the at least one saccharide raw material through phosphorolytic cleavage by the at least one alpha-phosphorylase and the at least one phosphorous source into an alpha-D-glucose 1-phosphate intermediate and a co-product; b) the conversion of the alpha-D-glucose 1-phosphate intermediate and the glucose substrate into trehalose by the at least one trehalose phosphorylase; c) the conversion of the fructose co-product into a glucose substrate by the at least one glucose isomerase.

    4. The method of claim 3, wherein the method is further characterized as a) a two-enzyme process, involving the reacting and mixing of the at least one alpha-phosphorylase, the at least one trehalose phosphorylase, the at least one saccharide raw material, the glucose substrate, and the at least one phosphorus source; and/or b) a three-enzyme process, involving the reacting and mixing of the at least one alpha-phosphorylase, the at least one trehalose phosphorylase, the at least one glucose isomerase, the at least one saccharide raw material, and the at least one phosphorus source; and/or c) a three-enzyme process, involving the reacting and mixing of the at least one alpha-phosphorylase, the at least one trehalose phosphorylase, the at least one glucose isomerase, the glucose substrate, the at least one saccharide raw material, and the at least one phosphorus source.

    5. The method of claim 1, wherein the method is carried out at a temperature of at least 30 C. up to 80 C.

    6. The method of claim 1, wherein the at least one saccharide raw material is mixed and reacted in the form of sucrose, preferably wherein the sucrose is added to the reaction in a concentration range of from 100 mM up to 2000 mM.

    7. The method of claim 1, wherein the at least one trehalose phosphorylase is characterized by one or more of the characteristics selected from the group consisting of the characteristics (A), (B), (C), and (D): (A) a thermal stability, wherein the thermal stability is characterized by a residual enzymatic activity of from 30% to 100% after incubation of the enzyme at 52 C. for 15 minutes; and/or (B) a thermal stability, wherein the thermal stability is characterized by a Tm50-value of at least 52 C. after incubation of the enzyme at 52 C. for 15 minutes; and/or (C) a thermal stability, wherein the thermal stability is characterized by a Tm50-value of between 52 C. and 90 C. after incubating the enzyme at 52 C. for 15 minutes; and/or (D) a thermal stability, wherein the thermal stability is in the form of a process stability characterized by an half-life of from 3 hours to 9 days or more.

    8. The method of claim 1, wherein the method is characterized by the use of an at least one trehalose phosphorylase which enables (i) a three-enzyme process comprising the steps of mixing and reacting, in any order, at least one alpha-phosphorylase, at least one glucose isomerase and at least one trehalose phosphorylase (TP), wherein the Productivity per kU of TP enzyme of trehalose from sucrose is at least 1.0 g/(L*h) per kU TP to 100 g/(L*h) per kU TP; and/or (ii) a two-enzyme process comprising the steps of mixing and reacting, in any order, a glucose substrate, at least one alpha-phosphorylase, and at least one trehalose phosphorylase (TP) without the addition of a glucose isomerase, wherein the Productivity per kU of TP enzyme of trehalose from saccharide raw material is at least 3 g/(L*h) per kU TP to 100 g/(L*h) per kU TP.

    9. The method of claim 1, wherein the at least one trehalose phosphorylase is a trehalose phosphorylase variant, wherein the variant comprises or consists of an amino acid sequence, wherein the amino acid sequence is at least 20% homologous and/or identical to the amino acid sequence of SEQ NO: 1, wherein the variant comprises two up to 15, three up to 15, four up to 15, five up to 15, six up to 15, seven up to 15, eight up to 15, nine up to 15, ten up to 15, eleven up to 15, twelve up to 15, thirteen up to 15, fourteen up to 15, or fifteen of the following amino acid positions selected from the group consisting of a) 712A, 712G, 712I, 712M, 712P or 712V, and/or b) 383A, 383G, 383I, 383L, 383M, 383V, 383N, 383C, 383Q, 383S or 383T, and/or c) 114A, 114G, 114I, 114M, 114P or 114V, and/or d) 118 is 118A, 118G, 118I, 118L, 118M, 118P or 118V, and/or e) 225A, 225G, 225I, 225L, 225M, 225P or 225V, and/or f) 304G, 304I, 304L, 304M, 304P or 304V, and/or g) 323A, 323G, 323I, 323L, 323M, 323P, or 323V, and/or h) 349W or 349Y, and/or i) 357A, 357I, 357L, 357M, 357P or 357V, and/or j) 487A, 487G, 487I, 487L, 487M, 487P or 487V, and/or k) 550A, 550G, 550I, 550L, 550M or 550P, and/or l) 556N, 556C, 556Q or S556T, and/or m) 564D or 564E, and/or n) 590N, 590C, 590Q, 590S, 590T, 590A, 590G, 590I, 590L, 590M, 590P or 590V, and/or o) 649D or 649E, wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1.

    10. The method of claim 1, wherein the at least one trehalose phosphorylase is a trehalose phosphorylase variant, wherein the variant comprises or consists of an amino acid sequence, wherein the amino acid sequence is at least 77% homologous and/or identical to the amino acid sequence of SEQ NO: 1, wherein the variant comprises an amino acid substitution at two or more of the following amino acid positions selected from the group consisting of amino acid positions 383, 114, 225, 304, 323, 349, 357, 550, 556, 564, and 649, wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1.

    11. The method of claim 10, wherein the trehalose phosphorylase variant comprises at least one further amino acid substitution at an amino acid position selected from the group consisting of amino acid positions 712, 118, 487 and 590, wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1

    12. The method of claim 1, wherein the at least one trehalose phosphorylase is a trehalose phosphorylase variant, wherein the variant comprises or consists of an amino acid sequence, wherein the amino acid sequence is at least 68% homologous and/or identical to the amino acid sequence of SEQ NO: 1, wherein the variant comprises an amino acid substitution at two or more of the following amino acid positions selected from the group consisting of amino acid positions 383, 114, 225, 304, 323, 349, 357, 550, 556, and 564, wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1.

    13. The method of claim 12, wherein the trehalose phosphorylase variant comprises at least one further amino acid substitution at an amino acid position selected from the group consisting of amino acid positions 712, 118, 487, 590, and 649, wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1

    14. The method of claim 1, wherein the at least one trehalose phosphorylase is a trehalose phosphorylase variant, wherein the variant comprises or consists of an amino acid sequence, wherein the amino acid sequence is at least 63% homologous and/or identical to the amino acid sequence of SEQ NO: 1, wherein the variant comprises an amino acid substitution at two or more of the following amino acid positions selected from the group consisting of amino acid positions 383, 114, 225, 304, 323, 349, 357, 556, and 564, wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1.

    15. The method of any of claim 14, wherein the trehalose phosphorylase variant comprises at least one further amino acid substitution at an amino acid positions selected from the group consisting of amino acid positions 712, 118, 487, 550, 590, and 649, wherein the amino acid numbering refers to an aligning position in SEQ ID NO: 1

    16. The method of claim 9, wherein the at least one trehalose phosphorylase variant is characterized by one or more of the characteristics selected from the group consisting of the characteristics (A), (B), (C), and (D): (A) a thermal stability, wherein the thermal stability is characterized by a residual enzymatic activity of from 30% to 100% after incubation of the enzyme at 52 C. for 15 minutes; and/or (B) a thermal stability, wherein the thermal stability is characterized by a Tm50-value of at least 52 C. after incubation of the enzyme at 52 C. for 15 minutes; and/or (C) a thermal stability, wherein the thermal stability is characterized by a Tm50-value of between 52 C. and 90 C. after incubating the enzyme at 52 C. for 15 minutes; and/or (D) a thermal stability, wherein the thermal stability is in the form of a process stability characterized by a half-life of from 3 hours to 9 days or more.

    17. The method of claim 9, wherein the at least one trehalose phosphorylase is characterized in that it (i) enables a Productivity per kU of TP enzyme of trehalose from the saccharide raw material sucrose of at least 1.0 g/(L*h) per kU TP to 100 g/(L*h) per kU TP; and/or (ii) enables a three-enzyme process comprising the steps of mixing and reacting, in any order, at least one alpha-phosphorylase, at least one glucose isomerase and at least one trehalose phosphorylase (TP), wherein the Productivity per kU of TP enzyme of trehalose from sucrose is at least 1.0 g/(L*h) per kU TP to 100 g/(L*h) per kU TP; and/or (iii) enables a two-enzyme process comprising the steps of mixing and reacting, in any order, a glucose substrate, at least one alpha-phosphorylase, and at least one trehalose phosphorylase (TP) without the addition of a glucose isomerase, wherein the Productivity per kU of TP enzyme of trehalose from saccharide raw material is at least 3 g/(L*h) per kU TP to 100 g/(L*h) per kU TP.

    18. The method of claim 1, wherein the at least one trehalose phosphorylase comprises or consists of an amino acid sequence, wherein the amino acid sequence is at least 80% identical and/or at least 80% homologous to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at one or more amino acid positions, wherein the one or more amino acid positions is/are selected from the group consisting of the amino acid positions 712, 383, 114, 118, 192, 197, 220, 225, 304, 306, 318, 323, 339, 349, 357, 459, 476, 481, 484, 487, 488, 506, 511, 526, 530, 532, 533, 537, 550, 556, 564, 590, 667, 703 and 705 of SEQ ID NO: 1.

    19. The method of claim 18, wherein the at least one trehalose phosphorylase is characterized by one or more of the characteristics selected from the group consisting of the characteristics (A), (B), (C), and (D): (A) a thermal stability, wherein the thermal stability is characterized by a residual enzymatic activity of from 30% to 100% after incubation of the enzyme at 52 C. for 15 minutes; and/or (B) a thermal stability, wherein the thermal stability is characterized by a Tm50-value of at least 52 C. after incubation of the enzyme at 52 C. for 15 minutes; and/or (C) a thermal stability, wherein the thermal stability is characterized by a Tm50-value of between 52 C. and 90 C. after incubating the enzyme at 52 C. for 15 minutes; and/or (D) a thermal stability, wherein the thermal stability is in the form of a process stability characterized by an half-life of from 3 hours to 9 days or more.

    20. The method of claim 18, wherein the at least one trehalose phosphorylase is characterized in that it (i) enables a Productivity per kU of TP enzyme of trehalose from the saccharide raw material sucrose of at least 1.0 g/(L*h) per kU TP to 100 g/(L*h) per kU TP; and/or (ii) enables a three-enzyme process comprising the steps of mixing and reacting, in any order, at least one alpha-phosphorylase, at least one glucose isomerase and at least one trehalose phosphorylase (TP), wherein the Productivity per kU of TP enzyme of trehalose from sucrose is at least 1.0 g/(L*h) per kU TP to 100 g/(L*h) per kU TP; and/or (iii) enables a two-enzyme process comprising the steps of mixing and reacting, in any order, a glucose substrate, at least one alpha-phosphorylase, and at least one trehalose phosphorylase (TP) without the addition of a glucose isomerase, wherein the Productivity per kU of TP enzyme of trehalose from saccharide raw material is at least 3 g/(L*h) per kU TP to 100 g/(L*h) per kU TP.

    21. The method of claim 1, wherein the at least one trehalose phosphorylase comprises or consists of an amino acid sequence, wherein the amino acid sequence is at least 85% identical and/or at least 85% homologous to the amino acid sequence of SEQ ID NO: 81 and/or SEQ ID NO: 160, wherein the amino acid sequence of the trehalose phosphorylase comprises an amino acid substitution at one or more amino acid positions, wherein the one or more amino acid positions is/are selected from the group consisting of amino acid positions 108, 112, 221, 300, 319, 345, 379, 483, 544, 550, 558, 584, 643, and 707 of SEQ ID NO: 81 or SEQ ID NO: 160.

    22. The method of claim 21, wherein the at least one trehalose phosphorylase is characterized by one or more of the characteristics selected from the group consisting of the characteristics (A), (B), (C), and (D): (A) a thermal stability, wherein the thermal stability is characterized by a residual enzymatic activity of from 30% to 100% after incubation of the enzyme at 52 C. for 15 minutes; and/or (B) a thermal stability, wherein the thermal stability is characterized by a Tm50-value of at least 52 C. after incubation of the enzyme at 52 C. for 15 minutes; and/or (C) a thermal stability, wherein the thermal stability is characterized by a Tm50-value of between 52 C. and 90 C. after incubating the enzyme at 52 C. for 15 minutes; and/or (D) a thermal stability, wherein the thermal stability is in the form of a process stability characterized by an half-life of from 3 hours to 9 days or more.

    23. The method of claim 21, wherein the at least one trehalose phosphorylase is characterized in that it (i) enables a Productivity per kU of TP enzyme of trehalose from the saccharide raw material sucrose of at least 1.0 g/(L*h) per kU TP to 100 g/(L*h) per kU TP; and/or (ii) enables a three-enzyme process comprising the steps of mixing and reacting, in any order, at least one alpha-phosphorylase, at least one glucose isomerase and at least one trehalose phosphorylase (TP), wherein the Productivity per kU of TP enzyme of trehalose from sucrose is at least 1.0 g/(L*h) per kU TP to 100 g/(L*h) per kU TP; and/or (iii) enables a two-enzyme process comprising the steps of mixing and reacting, in any order, a glucose substrate, at least one alpha-phosphorylase, and at least one trehalose phosphorylase (TP) without the addition of a glucose isomerase, wherein the Productivity per kU of TP enzyme of trehalose from saccharide raw material is at least 3 g/(L*h) per kU TP to 100 g/(L*h) per kU TP.

    24. The method of claim 1, wherein the at least one trehalose phosphorylase comprises or consists of an amino acid sequence, wherein the amino acid sequences is selected from the group consisting of any one of a) SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, and SEQ ID NO: 190; and/or b) SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159 and SEQ ID NO: 190; and/or c) SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, and SEQ ID NO: 189; and/or d) SEQ ID NO: 44, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 78, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 104, SEQ ID NO: 115, SEQ ID NO: 121, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 147, SEQ ID NO: 180, and SEQ ID NO: 188.

    25. The method of claim 1, wherein the at least one trehalose phosphorylase comprises or consists of an amino acid sequence selected from the group consisting of any one of SEQ ID NO: 2, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, and SEQ ID NO: 170.

    26. The method of claim 1, wherein the at least one alpha-phosphorylase is a sucrose phosphorylase, wherein the sucrose phosphorylase has at least 75% sequence identity and/or sequence homology to an amino acid sequence selected from the group consisting of SEQ ID NO: 202 and SEQ ID NO: 205.

    27. The method of claim 1, wherein the at least one glucose isomerase is a glucose isomerase with at least 95% sequence identity and/or sequence homology to the amino acid sequence of SEQ ID NO: 220.

    28. The method of claim 1, wherein i) the at least one alpha-phosphorylase is in form of a sucrose phosphorylase comprising or consisting of an amino acid sequence selected from the group of sequences consisting of SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218 and SEQ ID NO: 219; and/or ii) the at least one trehalose phosphorylase is in form of a trehalose phosphorylase comprising or consisting of an amino acid sequence selected from the group of sequences consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190; and/or iii) the at least one glucose isomerase is in form of a glucose isomerase comprising or consisting of an amino acid sequence selected from the group of sequences consisting of SEQ ID NO: 220 to SEQ ID NO: 240.

    Description

    [0335] In an embodiment, if not indicated to the contrary any activity, enzymatic activity, phosphorolysis activity and synthesis activity displayed or to be displayed by the polypeptide, or by the trehalose phosphorylase variant, and, preferably any trehalose phosphorylase of the pre nt invention is defined and, respectively, determined by the methods and assays, respectively, disclosed herein. The present invention is further illustrated by the figures, examples, tables and the sequence listing from which further features, embodiments and advantages may be taken, wherein:

    [0336] FIG. 1 is a diagram showing residual activity in % as a function of temperature for wild type trehalose phosphorylase of SEQ ID NO: 1 in the presence and in the absence of 1M sucrose added a stabilizing agent; and

    [0337] FIG. 2 is a diagram is a diagram showing residual activity in % as a function of time for wild type trehalose phosphorylase of SEQ ID NO: 1 and various trehalose phosphorylases of the invention.

    [0338]

    TABLE-US-00001 TABLE 1 Overview over Sequence IDs. Mutations (numbering refers to No of reference sequence specified SEQ ID Source mutations column 2) SEQ ID NO: 1 wild type, Schizophyllum None none commune, GenBank: ABC84380.1 SEQ ID NO: 2 variant of SEQ ID NO: 1 1 K705N SEQ ID NO: 3 variant of SEQ ID NO: 1 2 P383S, L712M SEQ ID NO: 4 variant of SEQ ID NO: 1 2 K705N, L712M SEQ ID NO: 5 variant of SEQ ID NO: 1 3 V10R, A506S, L712M SEQ ID NO: 6 variant of SEQ ID NO: 1 4 V10R, Y220F, A506S, L712M SEQ ID NO: 7 variant of SEQ ID NO: 1 4 V10R, L114I, Y220F, L712M SEQ ID NO: 8 variant of SEQ ID NO: 1 4 Y220F, A506S, K705N, L712M SEQ ID NO: 9 variant of SEQ ID NO: 1 6 V10R, L114I, Y220F, A506S, K705N, L712M SEQ ID NO: 10 variant of SEQ ID NO: 1 2 P383V, L712M SEQ ID NO: 11 variant of SEQ ID NO: 1 8 L114I, I118V, P383V, Q476G, K488A, A506S, A511S, L712M SEQ ID NO: 12 variant of SEQ ID NO: 1 9 L114I, I118V, A304I, F349Y, G357A, P383V, A5065, A511S, L712M SEQ ID NO: 13 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, F349Y, P383V, E481I, L712M SEQ ID NO: 14 variant of SEQ ID NO: 1 6 L114I, I118V, A304I, G357A, P383V, L712M SEQ ID NO: 15 variant of SEQ ID NO: 1 8 L114I, I118V, F349Y, G357A, P383V, E481I, K488A, L712M SEQ ID NO: 16 variant of SEQ ID NO: 1 8 L114I, I118V, A304I,I P383V, E481I, A506S, A511S, L712M SEQ ID NO: 17 variant of SEQ ID NO: 1 7 L114I, I118V, P383V, K488A, A506S, A511S, L712M SEQ ID NO: 18 variant of SEQ ID NO: 1 9 L114I, I118V, A304I, G357A, P383V, K488A, A506S A511S, L712M SEQ ID NO: 19 variant of SEQ ID NO: 1 5 L114I, I118V, P383V, E481I, L712M SEQ ID NO: 20 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, F349Y, P383V, K488A, L712M SEQ ID NO: 21 variant of SEQ ID NO: 1 7 L114I, I118V, P383V, E481I, A506S, A511S, L712M SEQ ID NO: 22 variant of SEQ ID NO: 1 7 L114I, I118V, S192V, A304I, G357A, P383V, L712M SEQ ID NO: 23 variant of SEQ ID NO: 1 7 L114I, I118V, S197G, A304I, G357A, P383V, L712M SEQ ID NO: 24 variant of SEQ ID NO: 1 7 L114I, I118V, N225V, A304I, G357A, P383V, L712M SEQ ID NO: 25 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, D306H, G357A, P383V, L712M SEQ ID NO: 26 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, P318H, G357A, P383V, L712M SEQ ID NO: 27 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, T323I, G357A, P383V, L712M SEQ ID NO: 28 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, L339I, G357A, P383V, L712M SEQ ID NO: 29 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, F349Y, G357A, P383V, L712M SEQ 1D NO: 30 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, A459S, L712M SEQ ID NO: 31 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, E481I, L712M SEQ ID NO: 32 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, A484S, L712M SEQ ID NO: 33 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, Q487V, L712M SEQ ID NO: 34 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, Q487A, L712M SEQ ID NO: 35 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, Q487L, L712M SEQ ID NO: 36 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, R526E, L712M SEQ ID NO: 37 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, E530V, L712M SEQ ID NO: 38 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, G532R, L712M SEQ ID NO: 39 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, D533G, L712M SEQ ID NO: 40 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, D537M, L712M SEQ ID NO: 41 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, V550I, L712M SEQ ID NO: 42 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, S556T, L712M SEQ ID NO: 43 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, T564E, L712M SEQ ID NO: 44 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, D590N, L712M SEQ ID NO: 45 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, D590A, L712M SEQ ID NO: 46 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, A649E, L712M SEQ ID NO: 47 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, R667E, L712M SEQ ID NO: 48 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, R667K, L712M SEQ ID NO: 49 variant of SEQ ID NO: 1 7 L114I, I118V, A304I, G357A, P383V, A703E, L712M SEQ ID NO: 50 variant of SEQ ID NO: 1 9 L114I, I118V, A304I, G357A, P383V, V550I, S556T, D590N, L712M SEQ ID NO: 51 variant of SEQ ID NO: 1 10 L114I, I118V, N225V, A304I, G357A, P383V, Q487L, T564E, D590N, L712M SEQ ID NO: 52 variant of SEQ ID NO: 1 10 L114I, I118V, A304I, T323I, G357A, P383V, V550I, T564E, D590N, L712M SEQ ID NO: 53 variant of SEQ ID NO: 1 10 L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487L, D590N, L712M SEQ ID NO: 54 variant of SEQ ID NO: 1 11 L114I, I118V, N225V, A304I, G357A, P383V, Q487L, V550I, S556T, D590N, L712M SEQ ID NO: 55 variant of SEQ ID NO: 1 11 L114I, I118V, N225V, A304I, G357A, P383V, Q487L, V550I, T564E, D590N, L712M SEQ ID NO: 56 variant of SEQ ID NO: 1 11 L114I, I118V, N225V, A304I, G357A, P383V, V550I, S556T, T564E, D590N, L712M SEQ ID NO: 57 variant of SEQ ID NO: 1 11 L114I, I118V, N225V, A304I, G357A, P383V, V550I, T564E, D590N, A649E, L712M SEQ ID NO: 58 variant of SEQ ID NO: 1 11 L114I, I118V, A304I, T323I, G357A, P383V, V550I, S556T, T564E, D590N, L712M SEQ ID NO: 59 variant of SEQ ID NO: 1 11 L114I, I118V, A304I, T323I, G357A, P383V, Q487L, V550I, D590N, A649E, L712M SEQ ID NO: 60 variant of SEQ ID NO: 1 12 L114I, I118V, N225V, A304I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, L712M SEQ ID NO: 61 variant of SEQ ID NO: 1 12 L114I, I118V, N225V, A304I, G357A, P383V, V550I, S556T, T564E, D590N, A649E, L712M SEQ ID NO: 62 variant of SEQ ID NO: 1 12 L114I, I118V, N225V, A3041, T323I, G357A, P383V, Q487L, V550I, T564E, D590N, L712M SEQ ID NO: 63 variant of SEQ ID NO: 1 12 L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487L, V550I, D590N, A649E, L712M SEQ ID NO: 64 variant of SEQ ID NO: 1 12 L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487A, V550I, S556T, V590N, L712M SEQ ID NO: 65 variant of SEQ ID NO: 1 12 L114I, I118V, N225V, A304I, T323I, G357A, P383V, V550I, S556I, T564E, D590N, L712M SEQ ID NO: 66 variant of SEQ ID NO: 1 12 L114I, I118V, A304I, T323I, F349Y, G357A, P383V, Q487L, V550I, D590N, A649E, L712M SEQ ID NO: 67 variant of SEQ ID NO; 1 12 L114I, I118V, A304I, T323I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, L712M SEQ ID NO: 68 variant of SEQ ID NO: 1 12 L114I, I118V, A304I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, A649E, L712M SEQ ID NO: 69 variant of SEQ ID NO: 1 12 L114I, I118V, A304I, G357A, P383V, Q487L, V550I, S556T, T564E, D590N, A649E, L712M SEQ ID NO: 70 variant of SEQ ID NO: 1 13 L114I, I118V, N225V, A304I, T323I, F349Y, G357A, P383V, V550I, S556T, D590N, A649E, L712M SEQ ID NO: 71 variant of SEQ ID NO: 1 13 L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487L, V550I, S556T, D590N, A649E, L712M SEQ ID NO: 72 variant of SEQ ID NO: 1 13 L114I, I118V, N225V, A304I, F349Y, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, L712M SEQ ID NO: 73 variant of SEQ ID NO: 1 13 L114I, I118V, N225V, A304I, F349Y, G357A, P383V, Q487L, V550I, T564E, D590N, A649E, L712M SEQ ID NO: 74 variant of SEQ ID NO: 1 13 L114I, I118V, N225V, A304I, G357A, P383V, Q487L, V550I, S556T, T564E, D590N, A649E, L712M SEQ ID NO: 75 variant of SEQ ID NO: 1 13 L114I, I118V, N225V, A304I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, A649E, L712M SEQ ID NO: 76 variant of SEQ ID NO: 1 13 L114I, I118V, A304I, T323I, G357A, P383V, Q487L, V550I, S556T, T564E, D590N, A649E, L712M SEQ ID NO: 77 variant of SEQ ID NO: 1 13 L114I, I118V, A304I, T323I G357A, P383V, Q487L, V550I, S556T, T564E, D590N, A649E, L712M SEQ ID NO: 78 variant of SEQ ID NO: 1 14 L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, A649E, L712M SEQ ID NO: 79 variant of SEQ ID NO: 1 14 L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487G, V550I, S556T, T564E, D590N, A649E, L712M SEQ ID NO: 80 wild type, Hypholoma none none sublateritium FD-334 SS-4, Genbank: KJA27491.1 SEQ ID NO: 81 wild type, Grifoia frondosa, none none Genbank: ADM15725 SEQ ID NO: 82 wild type, Pleurotus none none ostreatus, Genbank: KDQ33172.1 SEQ ID NO: 83 wild type, Lentinus sajor- none none caju, UniProtKB/Swiss-Prot: Q9UV63.1 SEQ ID NO: 84 variant of SEQ ID NO: 1 1 N225V SEQ ID NO: 85 variant of SEQ ID NO: 1 1 A304I SEQ ID NO: 86 variant of SEQ ID NO: 1 1 T323I SEQ ID NO: 87 variant of SEQ ID NO: 1 1 P383V SEQ ID NO: 88 variant of SEQ ID NO: 1 1 Q487A SEQ ID NO: 89 variant of SEQ ID NO: 1 1 S556T SEQ ID NO: 90 variant of SEQ ID NO: 1 1 T564E SEQ ID NO: 91 variant of SEQ ID NO: 1 1 D590N SEQ ID NO: 92 variant of SEQ ID NO: 1 1 N225I SEQ ID NO: 93 variant of SEQ ID NO: 1 1 N225L SEQ ID NO: 94 variant of SEQ ID NO: 1 1 N225M SEQ ID NO: 95 variant of SEQ ID NO: 1 1 A304L SEQ ID NO: 96 variant of SEQ ID NO: 1 1 T323V SEQ ID NO: 97 variant of SEQ ID NO: 1 1 P383A SEQ ID NO: 98 variant of SEQ ID NO: 1 1 P383G SEQ ID NO: 99 variant of SEQ ID NO: 1 1 P383M SEQ ID NO: 100 variant of SEQ ID NO: 1 1 P383N SEQ ID NO: 101 variant of SEQ ID NO: 1 1 P383C SEQ ID NO: 102 variant of SEQ ID NO: 1 1 P383Q SEQ ID NO: 103 variant of SEQ ID NO: 1 1 P383S SEQ ID NO: 104 variant of SEQ ID NO: 1 1 P383T SEQ ID NO: 105 variant of SEQ ID NO: 1 1 Q487G SEQ ID NO: 106 variant of SEQ ID NO: 1 1 Q487L SEQ ID NO: 107 variant of SEQ ID NO: 1 1 Q487M SEQ ID NO: 108 variant of SEQ ID NO: 1 1 V550P SEQ ID NO: 109 variant of SEQ ID NO: 1 1 D590G SEQ ID NO: 110 variant of SEQ ID NO: 1 2 I118V, P383V SEQ ID NO: 111 variant of SEQ ID NO: 1 2 I118V, S556T SEQ ID NO: 112 variant of SEQ ID NO: 1 2 I118V, T564E SEQ ID NO: 113 variant of SEQ ID NO: 1 2 I118V, D590N SEQ ID NO: 114 variant of SEQ ID NO: 1 2 N225V, A304I SEQ ID NO: 115 variant of SEQ ID NO: 1 2 N225V, P383V SEQ ID NO: 116 variant of SEQ ID NO: 1 2 N225V, Q487A SEQ ID NO: 117 vanant of SEQ ID NO: 1 2 N225V, V550I SEQ ID NO: 118 variant of SEQ ID NO: 1 2 N225V, S556T SEQ ID NO: 119 variant of SEQ ID NO: 1 2 N225V, S590N SEQ ID NO: 120 variant of SEQ ID NO: 1 2 A304I, T323I SEQ ID NO: 121 variant of SEQ ID NO: 1 2 A304I, P383V SEQ ID NO: 122 variant of SEQ ID NO: 1 2 A304I, Q487A SEQ ID NO: 123 variant of SEQ ID NO: 1 2 A304I, S556T SEQ ID NO: 124 variant of SEQ ID NO: 1 2 A304I, T564E SEQ ID NO: 125 variant of SEQ ID NO: 1 2 A304I, D590N SEQ ID NO: 126 variant of SEQ ID NO: 1 2 T323I, G357A SEQ ID NO: 127 variant of SEQ ID NO: 1 2 T323I, Q487A SEQ ID NO: 128 variant of SEQ ID NO: 1 2 T323I, S556T SEQ ID NO: 129 variant of SEQ ID NO: 1 2 T323I, T564E SEQ ID NO: 130 variant of SEQ ID NO: 1 2 T323I, D590N SEQ ID NO: 131 variant of SEQ ID NO: 1 2 T323I, A649E SEQ ID NO: 132 variant of SEQ ID NO: 1 2 F349Y, P383V SEQ ID NO: 133 variant of SEQ ID NO: 1 2 F349Y, D590N SEQ ID NO: 134 variant of SEQ ID NO: 1 2 G357A, P383V SEQ ID NO: 135 variant of SEQ ID NO: 1 2 G357A, D590N SEQ ID NO: 136 variant of SEQ ID NO: 1 2 P383V, Q487A SEQ ID NO: 137 variant of SEQ ID NO: 1 2 P383V, V550I SEQ ID NO: 138 variant of SEQ ID NO: 1 2 P383V, S556T SEQ ID NO: 139 variant of SEQ ID NO: 1 2 P383V, T564E SEQ ID NO: 140 variant of SEQ ID NO: 1 2 P383V, D590N SEQ ID NO: 141 variant of SEQ ID NO: 1 2 P383V, A649E SEQ ID NO: 142 variant of SEQ ID NO: 1 2 Q487A, T564E SEQ ID NO: 143 variant of SEQ ID NO: 1 2 Q487A, D590N SEQ ID NO: 144 variant of SEQ ID NO: 1 2 Q487A, A649E SEQ ID NO: 145 variant of SEQ ID NO: 1 2 V550I, D590N SEQ ID NO: 146 variant of SEQ ID NO: 1 2 S556T, T564E SEQ ID NO: 147 variant of SEQ ID NO: 1 2 S556T, D590N SEQ ID NO: 148 variant of SEQ ID NO: 1 2 S556T, A649E SEQ ID NO: 149 variant of SEQ ID NO: 1 2 T564E, D590N SEQ ID NO: 150 variant of SEQ ID NO: 1 2 T564E, L712M SEQ ID NO: 151 variant of SEQ ID NO: 1 2 D590N, A649E SEQ ID NO: 152 variant of SEQ ID NO: 1 2 D590N, L712M SEQ ID NO: 153 variant of SEQ ID NO: 1 2 A649E, L712M SEQ ID NO: 154 variant of SEQ ID NO: 1 13 L114I, N225V, A304I, T323I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, A649E, L712M SEQ ID NO: 155 variant of SEQ ID NO: 1 13 L114I, I118V, N225V, T323I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, A649E, L712M SEQ ID NO: 156 variant of SEQ ID NO: 1 13 L114I, I118V, N225V, A304I, T323I, P383V, Q487A, V550I, S5561, T564E, D590N, A649E, L712M SEQ ID NO: 157 variant of SEQ ID NO: 1 13 L114I, I118V, N225V, A304I, T323I, G357A, Q487A, V550I, S556T, T564E, D590N, A649E, L712M SEQ ID NO: 158 variant of SEQ ID NO: 1 13 L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487A, V550I, S556T, T564E, A649E, L712M SEQ ID NO: 159 variant of SEQ ID NO: 1 13 L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487A, V550I, S556T, T564E, D590N, A649E SEQ ID NO: 160 wild type, Grifola frondosa, none none UniProtKB/Swiss-Prot: O75003.1 SEQ ID NO: 161 variant of SEQ ID NO: 160 1 L108I SEQ ID NO: 162 variant of SEQ ID NO: 160 1 V112I SEQ ID NO: 163 variant of SEQ ID NO: 160 1 N221V SEQ ID NO: 164 variant of SEQ ID NO: 160 1 A300I SEQ ID NO: 166 variant of SEQ ID NO: 160 1 T319I SEQ ID NO: 166 variant of SEQ ID NO: 160 1 P379V SEQ ID NO: 167 variant of SEQ ID NO: 160 1 S550T SEQ ID NO: 168 variant of SEQ ID NO: 160 1 Q558E SEQ ID NO: 169 variant of SEQ ID NO: 160 1 A643E SEQ ID NO: 170 variant of SEQ ID NO: 160 1 L707M SEQ ID NO: 171 variant of SEQ ID NO: 160 2 L108I, N221V SEQ ID NO: 172 variant of SEQ ID NO: 160 2 L108I, A300I SEQ ID NO: 173 variant of SEQ ID NO: 160 2 L108I, T319I SEQ ID NO: 174 variant of SEQ ID NO: 160 2 L108I, V483A SEQ ID NO: 175 variant of SEQ ID NO: 160 2 L108I, S550T SEQ ID NO: 176 variant of SEQ ID NO: 160 2 P379V, L108I SEQ ID NO: 177 variant of SEQ ID NO: 160 2 P379V, V112I SEQ ID NO: 178 variant of SEQ ID NO: 160 2 P379V, N221V SEQ ID NO: 179 variant of SEQ ID NO: 160 2 P379V, A300I SEQ ID NO: 180 variant of SEQ ID NO: 160 2 P379V, T319I SEQ ID NO: 181 variant of SEQ ID NO: 160 2 P379V, F345Y SEQ ID NO: 182 variant of SEQ ID NO: 160 2 P379V, V483A SEQ ID NO: 183 variant of SEQ ID NO: 160 2 P379V, V544I SEQ ID NO: 184 variant of SEQ ID NO: 160 2 P379V, S550T SEQ ID NO: 185 variant of SEQ ID NO: 160 3 P379V, V483A, Q558E SEQ ID NO: 186 variant of SEQ ID NO: 160 3 P379V, V544I, S550T SEQ ID NO: 187 variant of SEQ ID NO: 160 3 P379V, V544I, Q558E SEQ ID NO: 188 variant of SEQ ID NO: 160 3 P379V, S550T, Q558E SEQ ID NO: 189 variant of SEQ ID NO: 160 4 P379V, V483A,V544I, Q558E SEQ ID NO: 190 variant of SEQ ID NO: 1 14 L114I, I118V, N225V, A304I, T323I, G357A, P383V, Q487L, V550I, S556T, T564E, D590N, A649E, L712M SEQ ID NO: 191 wild type, Schizophyllum none none commune H4-8, NCBI Reference Sequence: Xp_003035156.1 SEQ ID NO: 192 wild type, Trametes none none cinnabarine, Genbank: CDO74881.1 SEQ ID NO: 193 wild type, Hypsizygus none none marmoreus, Genbank: KYQ39707.1 SEQ ID NO: 194 wild type, Trametes none none versicolor FP-101664 SS1, NCBI Reference Sequence: XP_008036133.1 SEQ ID NO: 195 wild type, Pleurotus none none pulmonarius, UniProtKB/Swiss-Prot: A6YRN9.1 SEQ ID NO: 196 wild type, Agaricus bisporus none none var. bisporus H97, NCBI Reference Sequence: XP_006458503.1 SEQ ID NO: 197 wild type, Agaricus bisporus none none var. burnettii JB137-S8, NCBI Reference Sequence: XP_007326883.1 SEQ ID NO: 198 wild type, Laetiporus none none sulphureus 93-53, Genbank: KZT11205.1 SEQ ID NO: 199 wild type, Gloeophyllum none none trabeum ATCC 11539, NCBI Reference Sequence: XP_007863746.1 SEQ ID NO: 200 wild type, Grifola frondosa, none none Genbank: OBZ75413.1 SEQ ID NO: 201 wild type, Trametes none none pubescens, Genbank: OJT04097.1 SEQ ID NO: 202 wild type, Bifidobacterium none none magnum, NCBI Reference Sequence: WP_022860341.1 SEQ ID NO: 203 wild type, Bifidobacterium none none longum, GenBank: ADQ02266.1 SEQ ID NO: 204 wild type, Bifidobacterium none none animalis, NCBI Reference Sequence: WP_004218429.1 SEQ ID NO: 205 wild type, Bifidobacterium none none thermophilum, NCBI Reference Sequence: WP_015449645.1 SEQ ID NO: 206 wild type, Bifidobacterium none none adolescensis, NCBI Reference Sequence: WP_011742626.1 SEQ ID NO: 207 variant of SEQ ID NO: 202 1 E92L SEQ ID NO: 208 variant of SEQ ID NO: 202 1 S124Q SEQ ID NO: 209 variant of SEQ ID NO: 202 1 S124K SEQ ID NO: 210 variant of SEQ ID NO: 202 1 S124T SEQ ID NO: 211 variant of SEQ ID NO: 202 1 A148R SEQ ID NO: 212 variant of SEQ ID NO: 202 1 A148K SEQ ID NO: 213 variant of SEQ ID NO: 202 1 T157D SEQ ID NO: 214 variant of SEQ ID NO: 202 1 Q188Y SEQ ID NO: 215 variant of SEQ ID NO: 202 1 I231V SEQ ID NO: 216 variant of SEQ ID NO: 202 1 L371A SEQ ID NO: 217 variant of SEQ ID NO: 202 1 T461G SEQ ID NO: 218 variant of SEQ ID NO: 202 2 A148K, Q188Y SEQ ID NO: 219 variant of SEQ ID NO: 202 5 A148K, T157D, Q188Y, L371A, T461G SEQ ID NO: 220 wild type, Streptomyces sp. none none SK, PDB: 4HHL_A SEQ ID NO: 221 variant to SEQ ID NO: 220 1 A33I SEQ ID NO: 222 variant to SEQ ID NO: 220 1 A33N SEQ ID NO: 223 variant to SEQ ID NO: 220 1 L34F SEQ ID NO: 224 variant to SEQ ID NO: 220 1 D35C SEQ ID NO: 225 variant to SEQ ID NO: 220 1 F53L SEQ ID NO: 226 variant to SEQ ID NO: 220 1 A89V SEQ ID NO: 227 variant to SEQ ID NO: 220 1 T90S SEQ ID NO: 228 variant to SEQ ID NO: 220 1 T95R SEQ ID NO: 229 variant to SEQ ID NO: 220 1 T95Y SEQ ID NO: 230 variant to SEQ ID NO: 220 1 R10K SEQ ID NO: 231 variant to SEQ ID NO: 220 1 I59F SEQ ID NO: 232 variant to SEQ ID NO: 220 3 R10K, F53L, T95Y SEQ ID NO: 233 variant to SEQ ID NO: 220 4 R10K, F53L, T90S, T95Y SEQ ID NO: 234 variant to SEQ ID NO: 220 5 R10K, A33N, F53L, T90S, T95Y SEQ ID NO: 235 variant to SEQ ID NO: 220 3 R10K, F53L, T90S SEQ ID NO: 236 variant to SEQ ID NO: 220 4 R10K, A89V, T90S, T95Y SEQ ID NO: 237 variant to SEQ ID NO: 220 5 R10K, A33I, F53L, T90S, T95Y SEQ ID NO: 238 variant to SEQ ID NO: 220 5 R10K, D35S, F53L, T90S, T95Y SEQ ID NO: 239 variant to SEQ ID NO: 220 4 R10K, A33N, I59F, T90S SEQ ID NO: 240 variant to SEQ ID NO: 220 5 R10K, A33I, D358, I59F, T90S

    TABLE-US-00002 TABLE 2 Amino acids and corresponding positions after alignment with SEQ ID NO: 1 of different wild type Trehalose phosphorylases at the positions 114, 118, 225, 304, 323, 349, 383, 487, 550, 556, 564, 590, 649, 712 of SEQ ID NO: 1 (The alignment was done using Clustal omega (Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, Lopez R Nucleic acids research 2010 July, 38 Suppl: W695-9). Genbank/ SEQ ID Source Uniprot SEQ ID Schizophyllum ABC84380.1 postition 114 118 225 304 323 349 383 NO: 1 commune amino acid L I N A T F P SEQ ID Schizophyllum XP_003035156.1 postition 114 118 225 304 323 349 383 NO: 191 commune H4-8 amino acid L I N A T F P SEQ ID Hypholoma KJA27491.1 postition 70 74 183 262 281 307 341 NO: 80 sublateritium amino acid L V N A T F P FD-334 SS-4 SEQ ID Hypsizygus KYQ39707.1 postition 108 112 221 300 319 345 379 NO: 193 marmoreus amino acid L V N A T F P SEQ ID Trametes CDO74881.1 postition 2 6 115 194 213 239 273 NO: 192 cinnabarina amino acid P S N L T F P SEQ ID Grifola O75003.1 postition 108 112 221 300 319 345 379 NO: 160 frondosa amino acid L V N A T F P SEQ ID Grifola ADM15725.1 postition 108 112 221 300 319 345 379 NO: 81 frondosa amino acid L V N A T F P SEQ ID Pleurotus KDQ33172.1 postition 110 114 223 302 321 347 381 NO: 82 ostreatus PC15 amino acid L I N A T F P SEQ ID Pleurotus A6YRN9.1 postition 112 116 225 304 323 349 383 NO: 195 pulmonarius amino acid L I N A T F P SEQ ID Trametes XP_008036133.1 postition 111 115 224 303 322 348 382 NO: 194 versicolor amino acid L V N L T F P FP-101664 SS1 SEQ ID Agaricus XP_006458503.1 postition 110 114 223 302 321 347 381 NO: 196 bisporus var. amino acid L V N A T F P bisporus H97 SEQ ID Agaricus XP_007326883.1 postition 110 114 223 302 321 347 381 NO: 197 bisporus var. amino acid L V N A T F P burnettii JB137-S8 SEQ ID Laetiporus KZT11205.1 postition 108 112 221 300 319 345 380 NO: 198 sulphureus 93-53 amino acid L V N A T F P SEQ ID Lentinus Q9UV63.1 postition 112 116 225 304 323 349 383 NO: 83 sajor-caju amino acid L I N A T F P (Pleurotus sajor-caju) SEQ ID Gloeophyllum XP_007863746.1 postition 106 110 227 306 325 351 385 NO: 199 trabeum amino acid L V N L T F P ATCC 11539 SEQ ID Grifola OBZ75413.1 postition 108 112 221 300 319 345 379 NO: 200 frondosa amino acid L V N V T F P SEQ ID Trametes OJT04097.1 postition 70 74 183 263 297 NO: 201 pubescens amino acid L V N F P Genbank/ SEQ ID Source Uniprot SEQ ID Schizophyllum ABC84380.1 postition 487 550 556 564 590 649 712 NO: 1 commune amino acid Q V S T D A L SEQ ID Schizophyllum XP_003035156.1 postition 487 550 556 564 590 649 712 NO: 191 commune H4-8 amino acid Q V S T D V L SEQ ID Hypholoma KJA27491.1 postition 445 509 515 523 550 609 672 NO: 80 sublateritium amino acid T V S Q N A M FD-334 SS-4 SEQ ID Hypsizygus KYQ39707.1 postition 483 546 552 560 587 646 709 NO: 193 marmoreus amino acid A V S Q N A M SEQ ID Trametes CDO74881.1 postition 377 438 444 452 478 537 601 NO: 192 cinnabarina amino acid V V S Q N A L SEQ ID Grifola O75003.1 postition 483 544 550 558 584 643 707 NO: 160 frondosa amino acid V V S Q N A L SEQ ID Grifola ADM15725.1 postition 483 544 550 558 584 643 707 NO: 81 frondosa amino acid I V S Q N A L SEQ ID Pleurotus KDQ33172.1 postition 485 549 555 563 590 649 712 NO: 82 ostreatus PC15 amino acid A V S A N D L SEQ ID Pleurotus A6YRN9.1 postition 487 551 557 565 592 651 714 NO: 195 pulmonarius amino acid A V S A N D L SEQ ID Trametes XP_008036133.1 postition 486 547 553 561 587 646 710 NO: 194 versicolor amino acid V V S Q N A L FP-101664 SS1 SEQ ID Agaricus XP_006458503.1 postition 485 549 555 563 590 649 712 NO: 196 bisporus var. amino acid A V S Q N E L bisporus H97 SEQ ID Agaricus XP_007326883.1 postition 485 549 555 563 590 649 712 NO: 197 bisporus var. amino acid A V S Q N E L burnettii JB137-S8 SEQ ID Laetiporus KZT11205.1 postition 484 545 551 559 585 644 708 NO: 198 sulphureus 93-53 amino acid A V S A N E M SEQ ID Lentinus Q9UV63.1 postition 499 563 569 577 604 663 726 NO: 83 sajor-caju amino acid A V S A N D F (Pleurotus sajor-caju) SEQ ID Gloeophyllum XP_007863746.1 postition 489 552 558 566 592 651 715 NO: 199 trabeum amino acid V V S G N E L ATCC 11539 SEQ ID Grifola OBZ75413.1 postition 483 505 511 519 545 604 668 NO: 200 frondosa amino acid S V S Q N A L SEQ ID Trametes OJT04097.1 postition 401 462 468 476 502 561 625 NO: 201 pubescens amino acid V V S Q N A L

    EXAMPLES

    Example 1: Cloning of Wild Type Enzymes and Creation of Mutants

    [0339] Preparation of recombinant expression strains: All genes of the wild type enzymes, i.e. the trehalose phosphorylase gene from S. commune, the trehalose phosphorylase gene from G. frondosa, the sucrose phosphorylase gene from B. magnum and the glucose isomerase gene from Streptomyces sp. SK, were codon-optimized for expression in E. coli and synthesized by Eurofins MWG Operon or Geneart Thermo Fisher Scientific. The genes were cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmids were used for transformation of E. coli BL21 (DE3), of E. coli W3110 or of E. coli W3110 derivatives which carry certain deletions of endogenous enzyme genes (jointly referred to as Expression hosts).

    [0340] Molecular biology methods: Mutants of the enzymes were created by standard site-directed mutagenesis technologies as known in the state of the art.

    Example 2: Preparation of Enzyme Preparations

    [0341] Preparation of TP enzyme preparations: Expression hosts containing recombinant TPs were expressed in shaking flasks by inoculating Medium I (4.6 g/L yeast extract, 9.3 g/L peptone, 25 mM Na.sub.2HPO.sub.4*12 H.sub.2O, 25 mM KH.sub.2PO.sub.4, 50 mM NH.sub.4Cl.sub.2, Na.sub.2SO.sub.4, 5 g/L glycerol, 0.5 g/L glucose *1H.sub.2O, 2 mM MgSO.sub.4, 50 g/mL kanamycin) with a fresh overnight culture. Cultures were grown at 37 C. up to an optical density at 600 nm of 0.6-0.8. Cultures were induced with 0.1 mM IPTG final concentration. Expression was at 24-25 C. overnight. For the preparation of cell extract without sucrose cells were harvested by centrifugation and suspended in a buffer containing 50 mM potassium phosphate-buffer pH 7, 2 mM MgCl.sub.2, 0.5 mg/mL lysozyme and 20 U/mL nuclease. Cells were disrupted by sonication. Cell free extract containing soluble enzyme was separated from the debris by centrifugation. For the preparation of cell extract with sucrose as a stabilizing agent, cells were harvested by centrifugation and suspended in a buffer containing 100 mM potassium phosphate-buffer pH 7, 2 mM MgCl.sub.2, 0.5 mg/mL lysozyme and 20 U/mL nuclease. Cells were disrupted by sonication. Cell free extract containing soluble enzyme was separated from the debris by centrifugation and diluted 1:2 with 2 M sucrose solution.

    [0342] Preparation of SP enzyme preparations: Expression hosts containing recombinant SP was expressed by inoculating Medium I (4.6 g/L yeast extract, 9.3 g/L peptone, 25 mM Na.sub.2HPO.sub.4*12H.sub.2O, 25 mM KH.sub.2PO.sub.4, 50 mM NH.sub.4Cl.sub.2, Na.sub.2SO.sub.4, 5 g/L glycerol, 0.5 g/L glucose *1H.sub.2O, 2 mM MgSO.sub.4, 50 g/mL kanamycin) with a fresh overnight culture. Cultures were grown at 37 C. up to an optical density at 600 nm of 0.6-0.8. Cultures were induced with 0.1 mM IPTG final concentration. Expression was at 30 C. overnight. Preparation of cell free extract was done using procedures well known as described elsewhere. Cells were harvested by centrifugation and suspended in a buffer containing 50 mM potassium phosphate buffer pH 7, 2 mM MgCl.sub.2, 0.5 mg/mL lysozyme and 20 U/mL nuclease. Cell disruption was achieved by sonication or repeated freeze/thaw cycles. Cell free extract containing soluble enzyme was separated from the debris by centrifugation.

    [0343] Preparation of GI enzyme preparations: Expression hosts containing recombinant SP was expressed by inoculating Medium I (4.6 g/L yeast extract, 9.3 g/L peptone. 25 mM Na.sub.2HPO.sub.412H.sub.2O. 25 mM KH.sub.2PO.sub.4, 50 mM NH.sub.4Cl.sub.2, Na.sub.2SO.sub.4, 5 g/L glycerol; 0.5 g/L glucose *1H.sub.2O, 2 mM MgSO.sub.4, 50 g/mL kanamycin) with a fresh overnight culture. Cultures were grown at 37 C. up to an optical density at 600 nm of 0.8-1.0. Cultures were induced with 0.1 mM IPTG final concentration. Expression was at 30 C. overnight. Preparation of cell free extract was done by harvesting cells by centrifugation followed by chemoenzymatic lysis. For this, the cells were suspended in a buffer containing 50 mM potassium phosphate buffer pH 7, 1 CelLytic B Cell Lysis Reagent (Sigma), 2 mM Mg.sup.2+ (as MgCl.sub.2 or MgSO.sub.4), 0.5 mg/mL lysozyme and 20 U/mL nuclease, and incubated for 45 min at 30 C. Cell free extract containing soluble enzyme was separated from the debris by centrifugation for 30 min at 3.270g and 4C.

    Example 3: Effect of Sucrose on Thermal Stability

    [0344] 50 L aliquots of enzyme preparations of TP of SEQ ID NO: 1 from Example 2 with and without 1 M sucrose were incubated for 15 min at temperatures ranging from 36 to 53.7 C. Denatured protein was separated by centrifugation. The activity of the resulting supernatants as well as cell extract without a heat inactivation step was determined using the following Assay: Phosphorolytic activity was assayed at 30 C. using a continuous coupled assay in which the aG1P produced from trehalose is converted to glucose-6-phosphate by phosphoglucomutase. Glucose-6-phosphate and NADP is converted to 6-phospho-gluconate and NADPH by glucose 6-phosphate dehydrogenase. The detection is based on measuring the absorbance of NADPH at 340 nm. The assay solution contained: 75 mM potassium phosphate buffer pH 7. 2.5 mM NADP, 10 M glucose 1,6-bisphosphate. 10 mM MgCl.sub.2. 225 mM trehalose, 3 U/mL phosphoglucomutase and 3.4 U/mL glucose 6-phosphate dehydrogenase. 1 U is defined as the amount of enzyme that catalyzes the production of 1 mol of aG1P at the specified conditions. FIG. 1 is a denaturing profile of SEQ ID NO: 1 with and without 1 M sucrose as a stabilizing agent showing the obtained residual activities compared to the enzyme preparations without heat inactivation. The addition of 1 M sucrose results in an increase of Tm50 from approx. 40 C. to 47.5 C. 1 M sucrose was therefore chosen as a stabilizing agent for TP.

    Example 4: Residual Activity of TP Variants

    [0345] 50 L aliquots of enzyme preparations of TP enzymes of SEQ ID NO: 1-79; SEQ ID NO: 84-159 and SEQ ID NO: 190 from Example 2 with 1 M sucrose were incubated for 15 min at 52 C. Denatured protein was separated by centrifugation. The activity of the resulting supernatants as well as cell extract without a heat inactivation step s determined using the following Assay: Synthetic activity was assayed at 40 C. using the following conditions: 50 mM sodium ES buffer pH 7, 100 m aG1P and 500 mM glucose. Reaction progress was determined discontinuously by measuring liberated phosphate with an assay based on the complex formation with molybdate under acidic conditions. The molybdate complex is reduced by ferrous sulfate and yields a blue color, which is analyzed photometrically at 750 nm. For the analysis 250 L of sample are mixed with 250 L 0.5 M HCl and 500 L molybdate-reagent (73.2 g/L Fe(II)SO.sub.4*7H.sub.2O and 10 g/L ammonium molybdate*4H.sub.2O in 3.5% sulfuric acid). After incubation at RT for 15-30 min, absorbance is measured at 750 nm. The amount of inorganic phosphate in the sample is quantified using external standards. 1 U is d fined as the amount of enzyme that catalyzes the production of 1 mol of inorganic phosphate at the specified conditions. Residual activity was calculated by dividing the activity after the heat treatment by the activity without heat treatment and multiplication by 100. The resulting residual activities are listed in Table 3.

    TABLE-US-00003 TABLE 3 Residual activity in % after 15 min incubation at 52 C. residual activity in % after SEQ ID 15 min incubation at 52 C. SEQ ID NO: 1 19 SEQ ID NO: 2 30 SEQ ID NO: 3 64 SEQ ID NO: 4 30 SEQ ID NO: 5 39 SEQ ID NO: 6 54 SEQ ID NO: 7 55 SEQ ID NO: 8 42 SEQ ID NO: 9 63 SEQ ID NO: 10 68 SEQ ID NO: 11 39 SEQ ID NO: 12 55 SEQ ID NO: 13 61 SEQ ID NO: 14 30 SEQ ID NO: 15 75 SEQ ID NO: 16 43 SEQ ID NO: 17 50 SEQ ID NO: 18 41 SEQ ID NO: 19 48 SEQ ID NO: 20 75 SEQ ID NO: 21 52 SEQ ID NO: 22 37 SEQ ID NO: 23 36 SEQ ID NO: 24 47 SEQ ID NO: 25 41 SEQ ID NO: 26 45 SEQ ID NO: 27 42 SEQ ID NO: 28 31 SEQ ID NO: 29 55 SEQ ID NO: 30 38 SEQ ID NO: 31 33 SEQ ID NO: 32 39 SEQ ID NO: 33 31 SEQ ID NO: 34 53 SEQ ID NO: 35 51 SEQ ID NO: 36 34 SEQ ID NO: 37 37 SEQ ID NO: 38 39 SEQ ID NO: 39 35 SEQ ID NO: 40 46 SEQ ID NO: 41 51 SEQ ID NO: 42 43 SEQ ID NO: 43 42 SEQ ID NO: 44 55 SEQ ID NO: 45 32 SEQ ID NO: 46 43 SEQ ID NO: 47 41 SEQ ID NO: 48 35 SEQ ID NO: 49 53 SEQ ID NO: 50 96 SEQ ID NO: 51 106 SEQ ID NO: 52 99 SEQ ID NO: 53 105 SEQ ID NO: 54 105 SEQ ID NO: 55 77 SEQ ID NO: 56 106 SEQ ID NO: 57 84 SEQ ID NO: 58 88 SEQ ID NO: 59 86 SEQ ID NO: 60 113 SEQ ID NO: 61 117 SEQ ID NO: 62 101 SEQ ID NO: 63 79 SEQ ID NO: 64 101 SEQ ID NO: 65 99 SEQ ID NO: 66 105 SEQ ID NO: 67 99 SEQ ID NO: 68 92 SEQ ID NO: 69 104 SEQ ID NO: 70 76 SEQ ID NO: 71 97 SEQ ID NO: 72 90 SEQ ID NO: 73 105 SEQ ID NO: 74 108 SEQ ID NO: 75 107 SEQ ID NO: 76 97 SEQ ID NO: 78 109 SEQ ID NO: 79 97 SEQ ID NO: 84 29 SEQ ID NO: 85 23 SEQ ID NO: 86 28 SEQ ID NO: 87 38 SEQ ID NO: 88 38 SEQ ID NO: 89 42 SEQ ID NO: 90 28 SEQ ID NO: 91 40 SEQ ID NO: 92 25 SEQ ID NO: 93 35 SEQ ID NO: 94 31 SEQ ID NO: 95 35 SEQ ID NO: 96 29 SEQ ID NO: 97 38 SEQ ID NO: 98 64 SEQ ID NO: 99 22 SEQ ID NO: 100 22 SEQ ID NO: 101 54 SEQ ID NO: 102 26 SEQ ID NO: 103 41 SEQ ID NO: 104 78 SEQ ID NO: 105 33 SEQ ID NO: 106 29 SEQ ID NO: 107 30 SEQ ID NO: 108 26 SEQ ID NO: 109 33 SEQ ID NO: 110 56 SEQ ID NO: 111 43 SEQ ID NO: 112 25 SEQ ID NO: 113 63 SEQ ID NO: 114 32 SEQ ID NO: 115 72 SEQ ID NO: 116 24 SEQ ID NO: 117 37 SEQ ID NO: 118 25 SEQ ID NO: 119 32 SEQ ID NO: 120 29 SEQ ID NO: 121 72 SEQ ID NO: 122 27 SEQ ID NO: 123 32 SEQ ID NO: 124 27 SEQ ID NO: 125 55 SEQ ID NO: 126 25 SEQ ID NO: 127 23 SEQ ID NO: 128 30 SEQ ID NO: 129 23 SEQ ID NO: 130 51 SEQ ID NO: 131 31 SEQ ID NO: 132 64 SEQ ID NO: 133 67 SEQ ID NO: 134 61 SEQ ID NO: 135 59 SEQ ID NO: 136 63 SEQ ID NO: 137 56 SEQ ID NO: 138 70 SEQ ID NO: 139 62 SEQ ID NO: 140 69 SEQ ID NO: 141 62 SEQ ID NO: 142 26 SEQ ID NO: 143 28 SEQ ID NO: 144 23 SEQ ID NO: 145 29 SEQ ID NO: 146 34 SEQ ID NO: 147 55 SEQ ID NO: 148 26 SEQ ID NO: 149 43 SEQ ID NO: 150 34 SEQ ID NO: 151 40 SEQ ID NO: 152 56 SEQ ID NO: 153 26 SEQ ID NO: 154 81 SEQ ID NO: 155 68 SEQ ID NO: 156 94 SEQ ID NO: 157 69 SEQ ID NO: 158 74 SEQ ID NO: 159 87 SEQ ID NO: 190 100

    [0346] 50 L aliquots enzyme preparations of TP enzymes of SEQ ID 0: 160-189 from Example 2 with 1 M sucrose were incubated for 15 min at 52.5 C. Denatured protein was separated by centrifugation. The activity of the resulting supernatants as well as cell extract without a heat inactivation step was determined using the following Assay: Synthetic activity was assayed at 40 C. using the following conditions: 50 mM sodium MES buffer pH 7, 100 mM aG1P and 500 mM glucose. Reaction progress was determined discontinuously by measuring liberated phosphate with an a y based on the complex formation with molybdate under acidic conditions. The molybdate complex is reduced by ferrous sulfate and yields a blue color, which is analyzed photometrically at 750 nm. For the analysis 250 L of sample are mixed with 250 L 0.5 M HCl and 500 L molybdate-reagent (73.2 g/L Fe(II)SO.sub.4*7H.sub.2O and 10 g/L ammonium molybdate*4H.sub.2O in 3.5% sulfuric acid). After incubation at RT for 15-30 min, absorbance is measured at 750 nm. The amount of inorganic phosphate in the sample is quantified using external standards. 1 U is defined as the amount of enzyme that catalyzes the production of 1 mol of inorganic phosphate at the specified conditions. Residual activity was calculated by dividing the activity after the heat treatment by the activity without heat treatment and multiplication by 100. The resulting residual activities are listed in Table 4. As can be seen, the residual activity of SEQ ID NO: 176, 178, 180, 185 and SEQ ID NO: 188 are above 50% which means that the Tm50-values of these sequences are above 52.5 C.

    TABLE-US-00004 TABLE 4 Residual activity in % after 15 min incubation at 52 C. residual activity in % after 15 SEQ ID min incubation at 52.5 C. [%] SEQ ID NO: 160 9 SEQ ID NO: 161 14 SEQ ID NO: 162 22 SEQ ID NO: 163 33 SEQ ID NO: 164 26 SEQ ID NO: 165 37 SEQ ID NO: 166 37 SEQ ID NO: 167 20 SEQ ID NO: 168 34 SEQ ID NO: 169 12 SEQ ID NO: 170 15 SEQ ID NO: 171 37 SEQ ID NO: 172 29 SEQ ID NO: 173 42 SEQ ID NO: 174 12 SEQ ID NO: 175 25 SEQ ID NO: 176 57 SEQ ID NO: 177 42 SEQ ID NO: 178 56 SEQ ID NO: 179 38 SEQ ID NO: 180 74 SEQ ID NO: 181 16 SEQ ID NO: 182 28 SEQ ID NO: 183 21 SEQ ID NO: 184 43 SEQ ID NO: 185 58 SEQ ID NO. 186 42 SEQ ID NO: 187 49 SEQ ID NO: 188 70 SEQ ID NO: 189 41

    Example 5: Determination of Tm50 Values of TPs with Sucrose

    [0347] 50 L aliquots of enzyme preparations of TP enzymes of SEQ ID NO: 1, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 160, SEQ ID NO: 180 and SEQ ID NO: 188 from Example 2 with 1 M sucrose were incubated for 15 min at temperatures ranging from 36 to 53.7 C. Denatured protein was separated by centrifugation. The activity of the resulting supernatants as well as cell extract without a heat inactivation step was determined using the following Assay: Phosphorolytic activity was assayed at 30 C. using a continuous coupled assay in which the aG1P produced from trehalose is converted to glucose-6-phosphate by phosphoglucomutase. Glucose-6-phosphate and NADP is converted to 6-phospho-gluconate and NADPH by glucose 6-phosphate dehydrogenase. The detection is based on measuring the absorbance of NADPH at 340 nm. The assay solution contained: 75 mM potassium phosphate buffer pH 7, 2.5 mM NADP, 10 M glucose 1,6-bisphosphate, 10 mM MgCl.sub.2, 225 mM trehalose, 3 U/mL phosphoglucomutase and 3.4 U/mL glucose 6-phosphate dehydrogenase. 1 U is defined as the amount of enzyme that catalyzes the production of 1 mol of aG1P at the specified conditions. Residual activity was calculated by dividing the activity after the heat treatment by the activity without heat treatment and multiplication by 100. The Tm50-value was determined as the temperature at which the enzyme possesses 50% residual activity (Table 5).

    TABLE-US-00005 TABLE 5 Tm50-values of TP enzymes in 50 mM potassium phosphate buffer pH 7 containing 1 M sucrose SEQ ID Tm50 value SEQ ID NO: 1 47.5 SEQ ID NO: 44 52 SEQ ID NO: 50 53.5 SEQ ID NO: 54 54.5 SEQ ID NO: 57 54.5 SEQ ID NO: 58 54.5 SEQ ID NO: 62 56 SEQ ID NO: 64 56 SEQ ID NO: 65 56 SEQ ID NO: 71 56 SEQ ID NO: 72 56.5 SEQ ID NO: 73 57.5 SEQ ID NO: 74 56 SEQ ID NO: 78 56 SEQ ID NO: 79 56

    Example 6: Determination of Tm50 Values of SPs

    [0348] 50 L aliquots of enzyme preparations of SP enzymes of SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 218 and SEQ ID NO: 219 from Example 2 were incubated for 15 min at temperatures ranging from 50 C.80 C. After a regeneration step for 30 minutes on ice, and after centrifugation to remove precipitated protein, the sucrose phosphorylase activities in the supernatant were determined using the following assay: Phosphorolytic activity of a sucrose phosphorylase was assayed at 30 C. using a continuous coupled assay in which the aG1P produced from sucrose substrate is converted to glucose-6-phosphate by phosphoglucomutase. Glucose-6-phosphate and NADP is converted to 6-phospho-gluconate and NADPH by glucose 6-phosphate dehydrogenase. The detection is based on measuring the absorbance of NADPH at 340 nm. The assay solution contained: 75 mM potassium phosphate buffer pH 7, 2.5 mM NADP. 10 M glucose 1,6-bisphosphate, 10 mM MgCl.sub.2, 250 mM sucrose. 3 U/mL phosphoglucomutase and 3.4 U/mL glucose 6-phosphate dehydrogenase. 1 U is defined as the amount of enzyme that catalyzes the production of 1 mol of aG1P at the specified conditions. Additionally, the activity of cell extract without heat treatment was determined using the same assay. Residual activity was calculated by dividing the activity after the heat treatment by the activity without heat treatment and multiplication by 100. The Tm50-value was determined as the temperature at which the enzyme possesses 50% residual activity (Table 6).

    TABLE-US-00006 TABLE 6 Tm50-value of SP enzymes SEQ ID Tm50 value SEQ ID NO. 202 65 C. SEQ ID NO: 203 68 C. SEQ ID NO: 204 72 C. SEQ ID NO: 205 72 C. SEQ ID NO: 206 69 C. SEQ ID NO: 212 68.5 C. SEQ ID NO: 214 64.5 C. SEQ ID NO: 218 70 C. SEQ ID NO: 219 66.5 C.

    Example 7: Thermostability of GIs

    [0349] Denaturation profiles of glucose isomerase variants were determined by performing heat inactivation at different temperatures followed by activity measurements. Glucose isomerase preparations from Example 2 were divided into several aliquots. 60 L aliquots of each glucose isomerase variant was incubated at temperatures in the range 65-85 C. for 15 min. Denatured protein was separated by centrifugation for 10 min at 4 C. and 3,270g. The Activity of the supernatant was determined using the following assay: Glucose isomerase activity was assayed by monitoring the formation of glucose from fructose at 40 C. using the following conditions: 50 mM potassium phosphate buffer pH 7, 10 Mg.sup.2+ (as MgCl.sub.2 or MgSO.sub.4), 0.2 mL/mL reaction glucose isomerase enzyme preparations (diluted in 50 mM potassium phosphate buffer pH 7 so as to reach a maximum yield of 18%) and 200 mM fructose. The glucose produced from fructose by the action of glucose isomerase was determined using a discontinuous coupled assay in which the glucose is converted to glucose-6-phosphate by hexokinase. Glucose-6-phosphate and NADP is converted to 6-phospho-gluconate and NADPH by glucose 6-phosphate dehydrogenase. The detection is based on measuring the absorbance of NADPH at 340 nm. The D-GLUCOSEHK kit (HK/G6P-DH Format) was employed in the microplate format (product no. K-GLUHK-110A or K-GLUHK-220A available from Megazyme International Ireland, Wicklow, Ireland). The assay is performed according to the manufacturer recommendations and the amount of glucose in the sample is quantified using external standards. Another aliquot of each glucose isomerase variant was assayed directly for activity without heat-inactivation using the same assay. Residual activity was calculated by dividing the activity after the heat treatment by the activity without heat treatment and multiplication by 100. The Tm50-value was determined as the temperature at which the enzyme possesses 50% residual activity (Table 7).

    TABLE-US-00007 TABLE 7 Tm50-values of glucose isomerase variants in 50 mM potassium phosphate buffer pH 7 SEQ ID Tm50 value SEQ ID NO: 220 78 C. SEQ ID NO: 236 76 C.

    Example 8: Determination of Process Stability

    [0350] Process stability of the TPs of SEQ ID NO: 1, SEQ ID NO: 14, SEQ ID NO: 44, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 78, SEQ ID NO: 160, SEQ ID NO: 180 and SEQ ID NO: 188 was determined at 45 C. in 50 mM potassium phosphate buffer pH 7 containing 1M sucrose. Enzyme preparations with 1 M sucrose from Example 2 were diluted in 50 mM potassium phosphate buffer pH 7 containing 1M sucrose and incubated at 45 C. for 16 days. Samples were taken over time and the activity was measured using the following assay: Phosphorolytic activity was assayed at 30 C. using a continuous coupled assay in which the aG1P produced from trehalose is converted to glucose-6-phosphate by phosphoglucomutase. Glucose-6-phosphate and NADP is converted to 6-phospho-gluconate and NADPH by glucose 6-phosphate dehydrogenase. The detection is based on measuring the absorbance of NADPH at 340 nm. The assay solution contained: 75 mM potassium phosphate buffer pH 7, 2.5 mM NADP, 10 M glucose 1,6-bisphosphate. 10 mM MgCl.sub.2. 225 mM trehalose, 3 U/mL phosphoglucomutase and 3.4 U/mL glucose 6-phosphate dehydrogenase. 1 U is defined as the amount of enzyme that catalyzes the production of 1 mol of aG1P at the specified conditions. Residual activity was calculated by dividing the activity after the heat treatment by the activity without heat treatment and multiplication by 100. The results are shown in FIG. 2. SEQ ID NO: 1 showed a rapid activity loss within the first 3 hours and a half-life of approx. 1 hour. As expected from the Tm50-values, the new variants showed greatly improved process stability. SEQ ID NO: 62, SEQ ID NO: 65 and SEQ ID NO: 78 showed the highest improvements with half-lives of approx. 8.8 days. This constitutes an over 200-fold improvement compared to the wild-type enzyme.

    Example 9: Trehalose Synthesis: Three-Enzyme (3E) Process at Different Temperatures

    [0351] An SP enzyme is added to reaction aliquots containing 110 mM potassium phosphate buffer pH 7.5, 1.25 M sucrose, a GI enzyme and a TP enzyme preheated to 30 C., 45 C., 50 C. or 55 C., respectively. In order to avoid aG1P degrading side reactions, all enzymes are either heat purified (e.g. 15 min at 55 C. 60 C. or any other suitable temperature) obtained from a deletion strain, or purified using any biochemical method known in the art, such as precipitation or chromatography. Reactions are incubated at 30 C., 45 C. 50 C. or 55 C. at 500 rpm. SP enzymes may be selected from SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219: TP enzymes may be selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO; 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201; GI enzymes may be selected from SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240. Samples are taken over time and the amount of fructose, glucose, sucrose and trehalose is determined by HPLC (column: Luna 5 m NH.sub.2 100 , 2504.6 mm (Phenomenex), column temperature: 40 C., mobile phase: 80/20 (v/v) acetonitrile/water, isocratic analysis, flow rate: 2 mL/min, injection: 10 L. The amount of fructose, glucose, sucrose and trehalose in the sample is quantified using external standards). Alternatively, SP of SEQ ID NO: 10 or 11 and/or GI of SEQ ID NO: 11 can be used.

    Example 10: Trehalose Synthesis: Three-Enzyme (3E) Process at Different Sucrose Concentrations

    [0352] Similarly to Example 5, a SP enzyme is added to reaction aliquots containing 110 mM potassium phosphate buffer pH 7.5, a GI enzyme, a TP enzyme and either 300 mM or 1 M sucrose. In order to avoid aG1P degrading side reactions, all enzymes are either heat purified (e.g. 15 min at 55 C., 60 C. or any other suitable temperature) obtained from a deletion strain, or purified using any biochemical method known in the art, such as precipitation or chromatography. SP enzymes may be selected from SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219; TP enzymes may be selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO; 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105. SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122. SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201; GI enzymes may be selected from SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234. SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240. Reactions are incubated at 45 C. at 500 rpm. Samples are taken over time and the amount of fructose, glucose, sucrose and trehalose is determined by HPLC (column: Luna 5 m NH.sub.2 100 , 2504.6 mm (Phenomenex), column temperature: 40 C., mobile phase: 80/20 (v/v) acetonitrile/water, isocratic analysis, flow rate: 2 ml/min, injection: 10 L. The amount of fructose, glucose, sucrose and trehalose in the sample is quantified using external standards).

    Example 11: Trehalose Synthesis: Two-Enzyme (2E) Process

    [0353] An SP enzyme is added to reaction aliquots containing 110 mM potassium phosphate buffer pH 7.5, 300 mM sucrose, 300 mM glucose and a TP enzyme preheated to 30 C., 50 C. or 55 C., respectively. In order to avoid aG1P degrading side reactions, all enzymes are either heat purified (e.g. 15 min at 55 C., 60 C. or any other suitable temperature) obtained from a deletion strain, or purified using any biochemical method known in the art, such as precipitation or chromatography. Reactions are incubated at 30 C., 50 C. or 55 C. at 500 rpm. SP enzymes may be selected from SEQ ID NO: SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219; TP enzymes may be selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151. SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168. SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201. Samples are taken over time and the amount of fructose, glucose, sucrose and trehalose is determined by HPLC (column: Luna 5 m NH.sub.2 100 , 2504.6 mm (Phenomenex), column temperature: 40 C., mobile phase: 80/20 (v/v) acetonitrile/water, isocratic analysis, flow rate: 2 mL/min, injection: 10 L. The amount of fructose, glucose, sucrose and trehalose in the sample is quantified using external standards).

    Example 12: Determination of K.SUB.M.-Value

    [0354] Activity of glucose isomerase variants was determined at different fructose concentrations in the range 50-1000 mM fructose at the following conditions: 10 mM MgSO.sub.4, 50 mM potassium phosphate buffer pH 7.0. 40 C., using the following assay: The reaction for measuring glucose isomerase activity was conducted by monitoring the formation of glucose from fructose at following conditions: 50 mM potassium phosphate buffer pH 7, 10 mM MgSO.sub.4, 0.05 mL/mL reaction glucose isomerase enzyme preparations (diluted in 50 mM potassium phosphate buffer pH 7 so as to reach a maximum yield of 18%), 50-1000 mM fructose concentrations, and 40 C., The reaction was quenched by adding 0.1 mL 0.25 M HCl per mL reaction. The glucose produced from fructose by the action of glucose isomerase was determined using a discontinuous coupled assay in which glucose was converted to gluconolactone by glucose oxidase. Hydrogen peroxide, a by-product of this reaction, was used by horseradish peroxidase to oxidize 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), yielding a coloured product, which shows absorbance at 405 nm. A 10 L aliquot of acid-quenched reaction is mixed with 90 L of the assay mix containing 50 mM potassium phosphate buffer pH 6, 1 mM ABTS, 5 U/mL glucose oxidase and 1 U/mL horseradish peroxidase. After 60-70 min incubation at 30 C., the absorbance at 405 nm was measured (endpoint measurement). The amount of glucose in the sample is quantified using external standards. The resulting activities were fitted to the Michaelis-Menten equation from which a Michaelis constant K.sub.M for fructose for a given glucose isomerase variant was derived. As can be seen from Table 7, both tested variants show lower KM for fructose than the wild type glucose isomerase.

    TABLE-US-00008 TABLE 7 K.sub.M-value of glucose isomerase K.sub.M(fructose) SEQ ID [mM] SEQ ID NO: 220 237 SEQ ID NO: 236 152

    Example 13: Trehalose Synthesis: Three-Enzyme Process at 30 C., 39 C. and 40 C.

    [0355] Reaction aliquots containing potassium phosphate buffer pH 7.5, sucrose and MgSO.sub.4 were preheated to 30 C., 39 C. or 40 C., respectively, and SP, GI and TP enzyme preparations were added thereto (final concentrations: 200 mM surose, 10 mM MgSO.sub.4, 50 mM potassium phosphate buffer pH 7.5, 0.75 U/mL SP, 2.25 U/mL GI, 0.5 U/mL TP). The reaction containing TP enzyme preparation of SEQ ID NO: 160 had the following final composition: 200 mM surose, 10 mM MgSO.sub.4, 50 mM potassium phosphate buffer pH 7.5, 0.405 U/mL SP, 1,215 U/mL GI, 0.27 U/mL TP. In order to avoid aG1P degrading side reactions, all enzymes were either heat purified (15 min at 60 C.), obtained from a deletion strain, or purified using any biochemical method known in the art, such as precipitation or chromatography. The commercial GI Sternenzym GI 3000 (Sternenzym GmbH & Co. KG, Ahrensburg) was desalted using a PD-10 column from GE Healthcare according to the manufacturers instructions and eluted in 50 mM potassium phosphate buffer pH 7. The commercial immobilized GI Sweetzyme IT Extra (Novozymes, Sigma #G4166) was ground to a fine powder. Reactions were incubated with shaking at 500 rpm and at temperatures of 30 C., 39 C. or 40 C., respectively. Samples were taken after 23 h and the amount of trehalose was determined by HPLC (column: Luna 5 m NH.sub.2 100 , 2504.6 mm (Phenomenex), column temperature: 40 C., mobile phase: 80/20 (v/v) acetonitrile/water, isocratic analysis, flow rate: 2 mL/min, injection: 10 L), The amount of trehalose in the sample was quantified using external standards.

    [0356] The amount of trehalose formed and the Productivity per kU TP after 23 h at 30 C., 39 C. and 40 C. with different TP enzymes but using the same SP and GI enzyme are shown in Table 8.

    TABLE-US-00009 TABLE 8 Trehalose formation after 23 h at 30 C., 39 C. or 40 C. 30 C. 39 C. 40 C. Productivity Productivity Productivity 30 C. 39 C. 40 C. per kU TP per kU TP per kU TP Trehalose Trehalose Trehalose (g*L.sup.1* (g*L.sup.1* (g*L.sup.1* TP SP GI [mM] [mM] [mM] h.sup.1*kU.sup.1) h.sup.1*kU.sup.1) h.sup.1*kU.sup.1) SEQ ID SEQ ID Sternenzyme 28.5 0.0 0.0 0.9 0.0 0.0 NO: 1 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 21.4 56.0 24.3 0.7 1.8 0.8 NO: 87 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 27.1 32.9 n.d. 0.9 1.1 n.d. NO: 89 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 41.1 63.7 61.9 1.4 2.1 2.0 NO: 104 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 19.4 45.1 32.0 0.6 1.5 1.1 NO: 115 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 21.7 60.0 32.9 0.7 2.0 1.1 NO: 121 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 35.5 71.9 70.2 1.2 2.4 2.3 NO: 138 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 23.2 68.6 81.9 0.8 2.3 2.7 NO: 140 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 28.7 85.3 85.1 0.9 2.8 2.8 NO: 147 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 22.3 65.0 67.4 0.7 2.1 2.2 NO: 44 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 24.1 85.2 97.4 0.8 2.8 3.2 NO: 62 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 25.4 88.0 97.3 0.8 2.9 3.2 NO: 65 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 28.6 75.0 95.4 0.9 2.5 3.1 NO: 78 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 13.3 0.0 0.0 0.8 0.0 0.0 NO: 160 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 20.9 31.2 24.5 0.7 1.0 0.8 NO: 180 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 33.2 72.7 59.4 1.1 2.4 2.0 NO: 188 NO: 202 GI3000

    [0357] The data indicate that the use of TP variants is advantageous for trehalose synthesis in a trehalose synthesis process (three-enzyme process) with temperatures of 39C and 40 C. in comparison to wildtype sequences SEQ ID NO:1 and SEQ ID NO: 160.

    [0358] Table 9 shows the trehalose formation and the Productivity per kU TP after 23 h using different SP enzyme preparations together with either TP enzyme preparation of SEQ ID 1 or TP enzyme preparation of SEQ ID 65, respectively. In all assays, the same GI enzyme preparation has been added to the reaction.

    TABLE-US-00010 TABLE 9 Trehalose formation after 23 h at 30 C., 39 C. or 40 C. 30 C. 39 C. 40 C. Productivity Productivity Productivity 30 C. 39 C. 40 C. per kU TP per kU TP per kU TP Trehalose Trehalose Trehalose (g*L.sup.1* (g*L.sup.1* (g*L.sup.1* TP SP GI [mM] [mM] [mM] h.sup.1*kU.sup.1) h.sup.1*kU.sup.1) h.sup.1*kU.sup.1) SEQ ID SEQ ID Sternenzyme 28.5 0.0 0.0 0.9 0.0 0.0 NO: 1 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 31.2 0.0 0.0 1.0 0.0 0.0 NO: 1 NO: 218 GI3000 SEQ ID SEQ ID Sternenzyme 29.5 0.0 0.0 1.0 0.0 0.0 NO: 1 NO: 204 GI3000 SEQ ID SEQ ID Sternenzyme 29.1 0.0 0.0 1.0 0.0 0.0 NO: 1 NO: 205 GI3000 SEQ ID SEQ ID Sternenzyme 25.4 88.0 97.3 0.8 2.9 3.2 NO: 65 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 28.0 86.8 93.8 0.9 2.9 3.1 NO: 65 NO: 218 GI3000 SEQ ID SEQ ID Sternenzyme 26.2 87.9 94.1 0.9 2.9 3.1 NO: 65 NO: 204 GI3000 SEQ ID SEQ ID Sternenzyme 26.5 86.2 93.9 0.9 2.8 3.1 NO: 65 NO: 205 GI3000

    [0359] The data show that the advantages of producing trehalose by TP variants instead of wildtype enzymes are independent from the SP enzymes candidates present in the process. It is furthermore obvious, that different SP enzymes are suitable for trehalose synthesis.

    [0360] Table 10 shows the trehalose formation and the Productivity per kU TP after 23 h using different GI enzyme preparations together with either TP enzyme preparation of SEQ ID 1 or TP enzyme preparation of SEQ ID 65. In all assays, the same SP enzyme preparation has been added to the reaction.

    TABLE-US-00011 TABLE 10 Trehalose formation after 23 h at 30 C., 39 C. or 40 C. 30 C. 39 C. 40 C. Productivity Productivity Productivity 30 C. 39 C. 40 C. per kU TP per kU TP per kU TP Trehalose Trehalose Trehalose (g*L.sup.1* (g*L.sup.1* (g*L.sup.1* TP SP GI [mM] [mM] [mM] h.sup.1*kU.sup.1) h.sup.1*kU.sup.1) h.sup.1*kU.sup.1) SEQ ID SEQ ID Sternenzyme 28.5 0.0 0.0 0.9 0.0 0.0 NO: 1 NO: 202 GI3000 SEQ ID SEQ ID SEQ ID NO: 20.7 0.0 0.0 0.7 0.0 0.0 NO: 1 NO: 202 220 SEQ ID SEQ ID SEQ ID NO: 42.8 0.0 0.0 1.4 0.0 0.0 NO: 1 NO: 202 236 SEQ ID SEQ ID Novozymes 21.2 0.0 0.0 0.7 0.0 0.0 NO: 1 NO: 202 Sweetzyme SEQ ID SEQ ID Sternenzyme 25.4 88.0 97.3 0.8 2.9 3.2 NO: 65 NO: 202 GI3000 SEQ ID SEQ ID SEQ ID NO: 24.2 41.3 40.8 0.8 1.4 1.3 NO: 65 NO: 202 220 SEQ ID SEQ ID SEQ ID NO: 45.4 73.2 71.8 1.5 2.4 2.4 NO: 65 NO: 202 236 SEQ ID SEQ ID Novozymes 23.0 35.1 53.9 0.8 1.2 1.8 NO: 65 NO: 202 Sweetzyme

    [0361] The data show that the advantages of producing trehalose by TP variants instead of wildtype enzymes are independent from the GI enzyme candidates present in the process. It is furthermore obvious that different GI enzymes are suitable far trehalose synthesis.

    Example 14: Trehalose Synthesis: Three-Enzyme (3E) Process at 200 mM, 600 mM and 900 mM Sucrose

    [0362] Reaction aliquots containing potassium phosphate buffer pH 7.5, sucrose and MgSO.sub.4 were preheated to 30 C., and SP, GI and TP enzyme preparations were added thereto (final concentrations: 200 mM, 600 mM or 900 mM surose, respectively, 10 mM MgSO.sub.4, 50 mM potassium phosphate buffer pH 7.5, 0.525 U/mL SP, 1.575 U/mL GI, 0.35 U/mL TP). In order to avoid aG1P degrading side reactions, all enzymes were either heat purified (15 min at 60 C.), obtained from a deletion strain, or purified using any biochemical method known in the art, such as precipitation or chromatography. The commercial GI Sternenzym GI 3000 (Sternenzym GmbH & Co. KG, Ahrensburg) was desalted using a PD-10 column from GE Healthcare according to the manufacturers instructions and eluted in 50 mM potassium phosphate buffer pH 7. The commercial immobilized GI Sweetzyme IT Extra (Novozymes, Sigma #G4166) was ground to a fine powder. Reactions were incubated at 500 rpm at 30 C.

    [0363] Samples were taken over time and the amount of trehalose was determined by HPLC (column: Luna 5 m NH.sub.2 100 , 2504.6 mm (Phenomenex), column temperature: 40 C., mobile phase: 80/20 (v/v) acetonitrile/water, isocratic analysis, flow rate: 2 ml/min, injection: 10 L. The amount of trehalose in the sample was quantified using external standards). Results are shown in Table 11.

    TABLE-US-00012 TABLE 11 Trehalose formation after 23 h using 200, 600 or 900 mM sucrose. 200 mM 600 mM 900 mM sucrose sucrose sucrose 200 mM 600 mM 900 mM Productivity Productivity Productivity sucrose sucrase sucrose per kU TP per kU TP per kU TP Trehalose Trehalose Trehalose (g*L.sup.1* (g*L.sup.1* (g*L.sup.1* TP SP GI [mM] [mM] [mM] h.sup.1*kU.sup.1) h.sup.1*kU.sup.1) h.sup.1*kU.sup.1) SEQ ID SEQ ID Sternenzyme 22.9 22.8 23.3 1.08 1.07 1.10 NO: 1 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 23.4 25.5 25.0 1.10 1.20 1.18 NO: 1 NO: 218 GI3000 SEQ ID SEQ ID Sternenzyme 23.2 24.0 24.8 1.09 1.13 1.16 NO: 1 NO: 204 G13000 SEQ ID SEQ ID Sternenzyme 23.3 24.0 24.3 1.10 1.13 1.14 NO: 1 NO: 205 GI3000 SEQ ID SEQ ID Sternenzyme 18.2 19.6 20.0 0.86 0.92 0.94 NO: 87 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 23.5 31.3 28.1 1.11 1.47 1.32 NO: 104 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 14.2 15.2 15.1 0.67 0.72 0.71 NO: 115 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 21.1 23.4 26.1 0.99 1.10 1.23 NO: 121 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 25.5 30.8 28.6 1.20 1.45 1.34 NO: 138 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 20.6 23.0 21.7 0.97 1.08 1.02 NO: 140 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 21.0 22.3 21.9 0.99 1.05 1.03 NO: 147 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 17.5 18.2 16.5 0.82 0.86 0.77 NO: 44 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 21.7 19.7 16.9 1.02 0.93 0.79 NO: 62 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 22.2 23.4 21.0 1.04 1.10 0.99 NO: 65 NO: 202 GI3000 SEQ ID SEQ ID Sternenzyme 24.1 24.8 22.1 1.13 1.16 1.04 NO: 65 NO: 218 GI3000 SEQ ID SEQ ID Sternenzyme 23.9 23.4 20.3 1.12 1.10 0.96 NO: 65 NO: 204 GI3000 SEQ ID SEQ ID Sternenzyme 25.3 22.8 19.9 1.10 1.07 0.93 NO: 65 NO: 205 GI3000

    [0364] The data indicate that in the 3E trehalose synthesis process the TP and enzyme preparations do not show substrate inhibition up to 900 mM sucrose.

    Example 15: Trehalose Synthesis: Two-Enzyme (2E) Process at 30 C., 42 C. and 43 C.

    [0365] Reaction aliquots containing potassium phosphate buffer pH 7.5, sucrose, glucose and MgSO.sub.4 were preheated to 30 C., 42 C. or 43 C., respectively, and SP and TP enzymes were added thereto (final concentrations: 200 mM surose, 200 mM glucose, 10 mM MgSO.sub.4, 50 mM potassium phosphate buffer pH 7.5, 0.525 U/mL SP, 0.35 U/mL TP). In order to avoid aG1P degrading side reactions, all enzymes were either heat purified (15 min at 60 C.), obtained from a deletion strain, or purified using any biochemical method known in the art, such as precipitation or chromatography. The commercial GI Sternenzym GI 3000 (Sternenzym GmbH & Co. KG, Ahrensburg) was desalted using a PD-10 column from GE Healthcare according to the manufacturers instructions and eluted in 50 mM potassium phosphate buffer pH 7. Reactions were incubated at 500 rpm at 30 C., 42 C. or 43 C., respectively. Samples were taken over time and the amount of trehalose was determined by HPLC (column: Luna 5 m NH.sub.2 100 , 2504.6 mm (Phenomenex), column temperature: 40 C., mobile phase: 80120 (v/v) acetonitrile/water, isocratic analysis, flow rate: 2 mL/min, injection: 10 L. The amount of trehalose in the sample was quantified using external standards).

    [0366] Results are shown in Table 12.

    TABLE-US-00013 TABLE 12 Trehalose formation after 23 h at 30 C., 42 C. and 43 C. 30 C. 42 C. 43 C. Productivity Productivity Productivity 30 C. 42 C. 43 C. per kU TP per kU TP per kU TP Trehalose Trehalose Trehalose (g*L.sup.1* TP (g*L.sup.1* (g*L.sup.1* TP SP [mM] [mM] [mM] h.sup.1*kU.sup.1) h.sup.1*kU.sup.1) h.sup.1*kU.sup.1) SEQ ID SEQ ID 61.5 0.0 0.0 2.9 0.0 0.0 NO: 1 NO: 202 SEQ ID SEQ ID 121.4 117.5 101.8 5.7 5.5 4.8 NO: 104 NO: 202 SEQ ID SEQ ID 68.1 104.5 102.2 3.2 4.9 4.8 NO: 140 NO: 202 SEQ ID SEQ ID 64.8 106.7 108.6 3.0 5.0 5.1 NO: 62 NO: 202 SEQ ID SEQ ID 102.2 67.4 5.7 4.8 3.2 0.3 NO: 180 NO: 202

    [0367] The data indicate that the use of TP variants is advantageous for trehalose synthesis in a trehalose synthesis process (2E) with temperatures of 42 C. and 43 C. in comparison to wildtype sequence SEQ ID NO: 1.